Immune Monitoring Laboratory at Dartmouth-Hitchcock Medical Center


CMS - 1.6.8 - Pouebo

Multiplexed Cytokines

Multiplex technologies allow detection of multiple biomarkers simultaneously.  These technologies are crucial for the complex task of deciphering disease-specific biomarker patterns.

DartLab offers the Luminex and the Meso Scale Discovery (MSD) platforms for multiplexed cytokine assays.  The MSD platform is preferable for serum and plasma samples, and the new U-plex plates allows for more customization.

The complex mixture of multiple antibodies, both bead associated and reporter conjugated is the limiting factor in extensive multiplexing: all antibodies need to be highly specific and sensitive to limit any cross-reactivity within the sample set.

The different sensitivities across kits can most likely be explained by the different antibodies used in the kits, as well as the differences in manufacturer-recommended incubation times for the samples and beads.

Luminex Assay Principle

The multiplex cytokine assay format differs from conventional ELISA in one significant way— the multiplex capture antibody is attached to a bead whereas the ELISA capture antibody is attached to the microplate well.  The technology uses 5.6 micron magnetic microspheres, which are internally dyed with red and infrared fluorophores of differing intensities. Each bead is given a unique number, or bead region, allowing differentiation of one bead from another. Beads covalently bound to different antibodies can be mixed in the same assay, utilizing a 96-well microplate format.

(from Viracor-IBT Laboratory's website)

At the completion of the sandwich immunoassay:

Luminex sandwich

beads are read in single-file by dual lasers for classification and quantification of each analyte using the Bio-Plex Array Reader (Bio-Rad Laboratories Inc., Hercules, CA).

The Bio-Plex array reader is a flow cytometry-based instrument that combines fluidics, 2 lasers, 4 detectors, and real-time digital signal processing to distinguish the 100 different sets of color-coded microspheres, each bearing an analyte-specific assay. The fluidics system of the array reader aligns the microspheres into single file as they enter a stream of sheath fluid and then enter a flow cell:


DartLab Luminex assay request form

Click here to download multiplex cytokine assay request form.


DartLab Protocols

BioPlex protocol

MilliPlex protocol

Millipore human 41plex measures:

EGF, Eotaxin, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1ra, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MCP-3, MDC (CCL22), MIP-1α, MIP-1β, RANTES, TGFα, TNF-α, TNF-β, VEGF, sCD40L


Millipore mouse 32plex measures:

Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1α, IL-1β, IL-2, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IP-10, KC-like, LIF, LIX, M-CSF, MCP-1, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α, VEGF


Radar plot showing the concentration of 42 cytokines (pg/ml; log scale) measured in human plasma using a Milliplex kit.  Radar plots can be made in Excel: select Charts/Other/Radar.


Methods (for publication)

Multiplex cytokine assays. Cytokines were measured using Millipore human cytokine multiplex kits (EMD Millipore.Corporation, Billerica, MA). Calibration curves from recombinant cytokine standards were prepared with threefold dilution steps in the same matrix as the samples.  High and low spikes (supernatants from stimulated human PBMCs and dendritic cells) were included to determine cytokine recovery.  Standards and spikes were measured in triplicate, samples were measured once, and blank values were subtracted from all readings. All assays were carried out directly in a 96-well filtration plate (Millipore, Billerica, MA) at room temperature and protected from light. Briefly, wells were pre-wet with 100 μl PBS containing 1% BSA, then beads together with a standard, sample, spikes, or blank were added in a final volume of 100 μl, and incubated together at room temperature for 30 min with continuous shaking. Beads were washed three times with 100 μl PBS containing 1% BSA and 0.05% Tween 20. A cocktail of biotinylated antibodies (50 μl/well) was added to beads for a further 30 min incubation with continuous shaking. Beads were washed three times, then streptavidin-PE was added for 10 min. Beads were again washed three times and resuspended in 125 μl of PBS containing 1% BSA and 0.05% Tween 20. The fluorescence intensity of the beads was measured in using the Bio-Plex array reader. Bio-Plex Manager software with five-parametric-curve fitting was used for data analysis.

How to acknowledge Shared Resources



Challenges in comparing results to those of traditional methods

Multiplex cytokine assay handbook - includes sample preparation

Selection of standards

Principles of curve fitting

Parameter logistics

Bio-Plex magnetic bead assay validation

Luminex extracellular assay protocol (from Invitrogen)

Assay performance of Bio-Plex human multiplex cytokine assay kits

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